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n engl j med 350;24 www.nejm.org june 10, 2004 The new england journal of medicine 2487 review article medical progress Pneumocystis Pneumonia Charles F. Thomas, Jr., M.D., and Andrew H. Limper, M.D. From the Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine, Department of Internal Medi- cine (C.F.T., A.H.L.), and the Department of Biochemistry and Molecular Biology (A.H.L.), Mayo Clinic College of Medicine, Rochester, Minn. Address reprint requests to Dr. Limper at 8-24 Stabile Bldg., Mayo Clinic, Rochester, MN 55905, or at limper. [email protected]. N Engl J Med 2004;350:2487-98. Copyright © 2004 Massachusetts Medical Society. neumocystis pneumonia remains the most prevalent opportun- istic infection in patients infected with the human immunodeficiency virus (HIV). 1,2 First identified as a protozoan nearly 100 years ago and reclassified as a fungus in 1988, pneumocystis cannot be propagated in culture. 3-6 Few treatment op- tions exist for patients with pneumocystis pneumonia. The number of patients who are receiving chronic immunosuppressive medication or who have an altered immune sys- tem and are thus at risk for pneumocystis pneumonia is rapidly growing. 2 Although the prevalence of the acquired immunodeficiency syndrome (AIDS) has decreased in the Western hemisphere owing to the routine use of highly active antiretroviral therapy (HAART), many patients worldwide do not have the resources for HAART. 1 The in- creased reservoir for infection among such patients leads to concern that resistance to trimethoprim–sulfamethoxazole, the most common treatment for pneumocystis pneu- monia, will emerge, although this has yet to happen. The application of molecular tech- niques to the study of pneumocystis has provided new insights into the complex cell biology of this fungus. In this article we summarize advances that have resulted from studies of the cell biology, biochemistry, and genetics of pneumocystis in the past several years and include recommendations for the diagnosis of pneumocystis pneu- monia, as well as for prophylaxis and treatment. Pneumocystis pneumonia is often the AIDS-defining illness in patients infected with HIV, occurring most frequently when the T-helper cell count (CD4+) is less than 200 cells per cubic millimeter. 7,8 Common symptoms of pneumocystis pneumonia include the subtle onset of progressive dyspnea, nonproductive cough, and low-grade fever. Acute dyspnea with pleuritic chest pain may indicate the development of a pneumotho- rax. Physical examination typically reveals tachypnea, tachycardia, and normal findings on lung auscultation. Patients with AIDS and pneumocystis pneumonia have a significantly increased number of pneumocystis organisms in their lungs, with fewer neutrophils, than do pa- tients with pneumocystis pneumonia in the absence of AIDS. 9 This greater organism burden results in a higher diagnostic yield of induced sputum and bronchoalveolar- lavage fluid to confirm pneumocystis pneumonia in patients with AIDS as compared with other conditions associated with immunosuppression. The smaller number of in- flammatory cells in patients with AIDS-related pneumocystis pneumonia also corre- lates with better oxygenation and survival as compared with pneumocystis pneumonia in patients without AIDS. 9 Patients with mild symptoms of pneumocystis pneumonia can often be treated as outpatients with the use of oral therapy and close follow-up. How- ever, patients with significant hypoxemia should be hospitalized for the administration of intravenous therapy. The development of worsening pneumonia with respiratory fail- p clinical features of pneumocystis pneumonia The New England Journal of Medicine Downloaded from nejm.org by JOHN VOGEL on March 19, 2014. For personal use only. No other uses without permission. Copyright © 2004 Massachusetts Medical Society. All rights reserved.

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n engl j med

350;24

www.nejm.org june

10, 2004

The

new england journal

of

medicine

2487

review article

medical progress

Pneumocystis Pneumonia

Charles F. Thomas, Jr., M.D., and

Andrew H. Limper, M.D.

From the Thoracic Diseases Research Unit,Division of Pulmonary and Critical CareMedicine, Department of Internal Medi-cine (C.F.T., A.H.L.), and the Departmentof Biochemistry and Molecular Biology(A.H.L.), Mayo Clinic College of Medicine,Rochester, Minn. Address reprint requeststo Dr. Limper at 8-24 Stabile Bldg., MayoClinic, Rochester, MN 55905, or at [email protected].

N Engl J Med 2004;350:2487-98.

Copyright © 2004 Massachusetts Medical Society.

neumocystis

pneumonia remains the most prevalent

opportun

-istic infection in patients infected with the human immunodeficiency virus(HIV).

1,2

First identified as a protozoan nearly 100 years ago and reclassified asa fungus in 1988, pneumocystis cannot be propagated in culture.

3-6

Few treatment op-tions exist for patients with pneumocystis pneumonia. The number of patients who arereceiving chronic immunosuppressive medication or who have an altered immune sys-tem and are thus at risk for pneumocystis pneumonia is rapidly growing.

2

Althoughthe prevalence of the acquired immunodeficiency syndrome (AIDS) has decreased inthe Western hemisphere owing to the routine use of highly active antiretroviral therapy(HAART), many patients worldwide do not have the resources for HAART.

1

The in-creased reservoir for infection among such patients leads to concern that resistance totrimethoprim–sulfamethoxazole, the most common treatment for pneumocystis pneu-monia

,

will emerge, although this has yet to happen. The application of molecular tech-niques to the study of pneumocystis has provided new insights into the complex cellbiology of this fungus. In this article we summarize advances that have resulted fromstudies of the cell biology, biochemistry, and genetics of pneumocystis in the pastseveral years and include recommendations for the diagnosis of pneumocystis pneu-monia, as well as for prophylaxis and treatment.

Pneumocystis pneumonia is often the AIDS-defining illness in patients infected withHIV, occurring most frequently when the T-helper cell count (CD4+) is less than 200cells per cubic millimeter.

7,8

Common symptoms of pneumocystis pneumonia includethe subtle onset of progressive dyspnea, nonproductive cough, and low-grade fever.Acute dyspnea with pleuritic chest pain may indicate the development of a pneumotho-rax. Physical examination typically reveals tachypnea, tachycardia, and normal findingson lung auscultation.

Patients with AIDS and pneumocystis pneumonia have a significantly increasednumber of pneumocystis organisms in their lungs, with fewer neutrophils, than do pa-tients with pneumocystis pneumonia in the absence of AIDS.

9

This greater organismburden results in a higher diagnostic yield of induced sputum and bronchoalveolar-lavage fluid to confirm pneumocystis pneumonia in patients with AIDS as comparedwith other conditions associated with immunosuppression. The smaller number of in-flammatory cells in patients with AIDS-related pneumocystis pneumonia

also corre-lates with better oxygenation and survival as compared with pneumocystis pneumoniain patients without AIDS.

9

Patients with mild symptoms of pneumocystis pneumoniacan often be treated as outpatients with the use of oral therapy and close follow-up. How-ever, patients with significant hypoxemia should be hospitalized for the administrationof intravenous therapy. The development of worsening pneumonia with respiratory fail-

p

clinical features of pneumocystis pneumonia

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ure in these patients is the most common reason foradmission to an intensive care unit. Among mostpatients with AIDS and pneumocystis pneumonia,the mortality rate is 10 to 20 percent during the ini-tial infection, but the rate increases substantiallywith the need for mechanical ventilation.

10

Patients who have pneumocystis pneumoniawithout AIDS typically present with an abrupt on-set of respiratory insufficiency that may correlatewith a tapered or increased dosage of immunosup-pressant medications.

11,12

Affected patients havemore neutrophils and fewer pneumocystis organ-isms in their lungs during an episode of pneu-mocystis pneumonia than do patients with AIDSwho have pneumocystis pneumonia.

9

The mortali-ty rate among patients with pneumocystis pneu-monia in the absence of AIDS is 30 to 60 percent,depending on the population at risk, with a great-er risk of death among patients with cancer thanamong patients undergoing transplantation orthose with connective-tissue disease.

2,13

Typical radiographic features of pneumocystispneumonia are bilateral perihilar interstitial infil-trates that become increasingly homogeneousand diffuse as the disease progresses (Fig. 1).

14

Less common findings include solitary or multi-ple nodules, upper-lobe infiltrates in patients re-ceiving aerosolized pentamidine, pneumatoceles,and pneumothorax. Pleural effusions and thorac-ic lymphadenopathy are rare. When chest radio-graphic findings are normal, high-resolution com-puted tomography, which is more sensitive thanchest radiography, may reveal extensive ground-glass attenuation or cystic lesions.

15

Pneumocystis pneumonia may be difficult to diag-nose owing to nonspecific symptoms and signs,the use of prophylactic drugs in the treatment ofHIV-infected patients, and simultaneous infectionwith multiple organisms (such as cytomegalovi-rus) in an immunocompromised host. The diagno-sis of pneumocystis pneumonia therefore requiresmicroscopical examination in order to identifypneumocystis from a clinically relevant source suchas specimens of sputum, bronchoalveolar fluid,or lung tissue, because pneumocystis cannot be cul-tured (Fig. 2).

16

Sputum induction with hypertonic saline has adiagnostic yield of 50 to 90 percent and should be

the initial procedure used to diagnose pneumocys-tis pneumonia, particularly in patients with AIDS.

17

If the initial specimen of induced sputum is neg-ative for pneumocystis, then bronchoscopy withbronchoalveolar lavage should be performed. Trans-bronchoscopic or surgical lung biopsy is rarelyneeded.

18

Trophic forms can be detected with mod-ified Papanicolaou, Wright–Giemsa, or Gram–Weigert stains. Cysts can be stained with Gomorimethenamine silver, cresyl echt violet, toluidineblue O, or calcofluor white. Monoclonal antibodiesfor detecting pneumocystis have a higher sensi-tivity and specificity in induced-sputum samplesthan conventional tinctorial stains have, but thedifference is much less in bronchoalveolar-lavagefluid.

19,20

An advantage of monoclonal antibodiesis their ability to stain both trophic forms and cysts,which is important because the trophic forms aregenerally more abundant during pneumocystispneumonia.

The use of the polymerase chain reaction (PCR)to detect pneumocystis nucleic acids has been anactive area of research. PCR has been shown tohave greater sensitivity and specificity for the diag-nosis of pneumocystis pneumonia from specimensof induced sputum and bronchoalveolar-lavagefluid than conventional staining when PCR prim-ers for the gene for pneumocystis mitochondriallarge-subunit ribosomal RNA (rRNA) are used.

21,22

In patients with positive PCR results in broncho-alveolar-lavage fluid or sputum but with negativesmears, clinical management of the disease remainsa challenge. However, we recommend treatment ofthese patients if immunosuppression is ongoing.

23

PCR testing of serum samples is not yet useful.

24

Although an elevated serum lactate dehydro-genase level has been noted in patients with pneu-mocystis pneumonia, it is likely to be a reflection ofthe underlying lung inflammation and injury rath-er than a specific marker for the disease.

25

Lowerlevels of plasma

S

-adenosylmethionine were ob-served in seven patients with confirmed pneu-mocystis pneumonia than in patients with otherpulmonary infections, but the usefulness of thistest will need to be confirmed in a larger cohort ofpatients.

26

Primary prophylaxis against pneumocystis pneu-monia in HIV-infected adults, including pregnant

diagnosis of pneumocystis infection

prophylaxis and treatment of pneumocystis pneumonia

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2489

women and patients receiving HAART, should be-gin when the CD4+ count is less than 200 cells permillimeter or if there is a history of oropharyngealcandidiasis.

8

Medications recommended for pro-phylaxis are listed in Table 1. Patients who havehad previous episodes of pneumocystis pneumo-nia should receive lifelong secondary prophylaxis,unless reconstitution of the immune system occursas a result of HAART. Primary or secondary pro-phylaxis should be discontinued in HIV-infected pa-tients who have had a response to HAART, as shownby an increase in the CD4+ cell count to more than200 cells per cubic millimeter for a period of threemonths. Prophylaxis should be reintroduced ifthe CD4+ count falls to less than 200 cells per cubicmillimeter.

Patients who are not infected with HIV but arereceiving immunosuppressive medications or whohave an underlying acquired or inherited immu-nodeficiency should receive prophylaxis againstpneumocystis pneumonia. In a retrospective se-ries, a corticosteroid dose that was the equivalentof 16 mg of prednisone or more for a period of eightweeks was associated with a significant risk ofpneumocystis pneumonia in patients who did nothave AIDS.

12

Similar observations have been notedin patients with cancer or with connective-tissuedisease that was treated with corticosteroids.

2,11

The notion that trimethoprim–sulfamethoxazoleis contraindicated for pneumocystis pneumoniaprophylaxis in patients treated with methotrexatemay be outdated, because in patients treated withup to 25 mg of methotrexate per week who re-ceived prophylaxis, severe myelosuppression didnot develop.

27

Such patients need to receive folatesupplementation (1 mg per day), or leucovorin onthe day after receiving methotrexate, and carefulmonitoring of the results of complete blood countsand liver-function tests is necessary.

Medications used to treat pneumocystis pneu-monia are listed in Table 2. Trimethoprim–sulfa-methoxazole is most effective in treating severepneumocystis pneumonia. Unfortunately, adverseeffects are common, and patients with known al-lergies to sulfa cannot tolerate this therapy. Corti-costeroids are of benefit in HIV-infected patientswith pneumocystis pneumonia who have hypox-emia (the partial pressure of arterial oxygen whilethe patient is breathing room air is under 70 mm Hgor the alveolar–arterial gradient is above 35). Suchpatients should receive prednisone at a dose of40 mg twice daily for five days, then 40 mg daily on

days 6 through 11, and then 20 mg daily on days 12though 21.

28

In patients without AIDS but with se-vere pneumocystis pneumonia, a dose of 60 mg ormore of prednisone daily resulted in a better out-come than lower doses of prednisone.

13

The transmission of pneumocystis is not fully un-derstood, nor has its environmental niche beenidentified. For decades, the theory of the reactiva-tion of latent pneumocystis infection — which heldthat pneumocystis remained latent within a personand caused disease when the immune system failed— was popular. Now there is evidence that person-to-person transmission is the most likely mode ofacquiring new infections, although acquisition fromenvironmental sources may also occur.

29

In addi-tion, people who are not infected may be asymp-

epidemiologic featuresof pneumocystis pneumonia

Figure 1. Posteroanterior Chest Radiograph of a 68-Year-Old Patient with Pneumocystis Pneumonia That Developed as a Consequence of Long-Term Corticosteroid Therapy for an Inflammatory Neuropathy.

Mixed alveolar and interstitial infiltrates are more prominent on the right side than on the left.

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tomatic carriers of pneumocystis.

30-35

Althoughthe results of studies in animals and humans favorairborne transmission, respiratory isolation for pa-tients with pneumocystis pneumonia is not cur-rently recommended.

The emerging resistance to trimethoprim–sul-famethoxazole is being investigated with the use ofmolecular techniques to study mutations in the di-hydropteroate synthase gene, which encodes theenzyme inhibited by dapsone and sulfamethoxa-zole. Several reports have implicated specific mu-tations in dihydropteroate synthase that are associ-ated with the failure of prophylaxis and treatment,an increase in the risk of death, and the selectionof the dihydropteroate synthase mutations by expo-sure of patients to sulfa-containing drugs.

36-38

Onestudy, however, found no association between tri-

methoprim–sulfamethoxazole and dihydropteroatesynthase mutations, treatment failure, or death.

39

Because most patients harboring pneumocystisthat contains dihydropteroate synthase mutationsstill have a response to treatment with trimetho-prim–sulfamethoxazole, further research is neces-sary to determine the importance of these mutationsand whether the geographic variation of pneu-mocystis infection and of other mutations contrib-ute when clinical treatment fails.

The full identification and classification of pneu-mocystis took many decades. Pneumocysti

s

organ-isms were first identified by Carlos Chagas in theearly 20th century, with the use of a guinea-pig mod-

the biology of the parasite

Figure 2. Detection of Pneumocystis Forms with the Use of Different Stains.

Panel A shows typical pneumocystis cyst forms in a bronchoalveolar-lavage specimen stained with Gomori methena-mine (¬100). Thick cyst walls and some intracystic bodies are evident. Wright–Giemsa staining can be used for rapid identification of trophic forms of the organisms within foamy exudates, as shown in Panel B (arrows), in bronchoalveo-lar-lavage fluid or induced sputum but usually requires a high organism burden and expertise in interpretation (¬100). Calcofluor white is a fungal cyst-wall stain that can be used for rapid confirmation of the presence of cyst forms, as shown in Panel C (¬400). Immunofluorescence staining, shown in Panel D, can sensitively and specifically identify both pneumocystis trophic forms (arrowheads) and cysts (arrows) (¬400).

A B

C D

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el of trypanosome infection, and subsequently byAntonio Carinii, in infected rat lungs.

4,5

Both in-vestigators believed they had identified new formsof trypanosomes. Several years later, however, theDelanoës recognized that Chagas and Carinii hadidentified a new species with a unique tropism forthe lung; hence, the new species was named

Pneu-mocystis carinii

.

3

Pneumocystis was initially misclas-sified as a protozoan on the basis of the morpho-logic features of the small trophic form, the largercyst form, the development of up to eight progenywithin the cyst, and the rupture of the cyst to re-lease new trophic forms. In 1988, an analysis of thesmall rRNA subunit from pneumocystis pneumo-nia established a phylogenetic linkage to the fungalkingdom, and all subsequent genomic informationhas corroborated pneumocystis’s home within theascomycetous fungi.

6

Pneumocystis organisms have been identifiedin virtually every mammalian species. In humans,serologic surveys have shown nearly universal se-ropositivity to pneumocystis in tested populationsby two years of age.

40

Pneumocystis organisms en-compass a family of organisms that have a range ofgenetic characteristics and that are host-specific.For example, the pneumocystis that infects humans,which was recently renamed

P. jirovecii,

cannot in-fect the rat, and vice versa.

41

The reason for thisstringent host specificity is unclear. The use of bi-nomial nomenclature for human pneumocystismay be of less importance currently, given that vi-sualization of the organism is required for the di-agnosis of pneumonia.

42,43

When PCR has beenapplied to pneumocystis pneumonia in humans,

only

P. jirovecii

has been found, and for that reason,specifying the name of the species will become im-portant only if additional species of pneumocystisare found to infect humans.

The major obstacle to studying pneumocystisis the inability to achieve sustained propagation ofthe organism outside the host lung.

16

Many inves-tigators have attempted to cultivate pneumocystisusing a variety of techniques, but none have beensuccessful. Cell-free systems that use media prep-arations for growing protozoa, bacteria, and fun-gi and tissue-culture systems in which a variety ofcell lines are used have not been successful. At-tempts to simulate the intraalveolar environmentand to supplement media with numerous additives,such as surfactant, lung homogenates, or variouschemical compounds, have also proved unhelpful.Until the organism can be cultivated in vitro, the in-fected-animal model of pneumocystis pneumoniawill remain the source of organisms for study.

Pneumocystis has a unique tropism for the lung,where it exists primarily as an alveolar pathogenwithout invading the host. In rare cases, pneumo-cystis disseminates in the setting of severe underly-ing immunosuppression or overwhelming infec-tion.

44

Microscopically, one can identify the smalltrophic forms (1 to 4 µm in diameter) and the larg-er cysts (8 µm in diameter). Electron microscopi-cal studies have shown three stages of precysts, theearly, intermediate, and late stages (Fig. 3).

45-47

Thetrophic forms are predominantly haploid, thoughdiploid forms also exist, whereas the cyst containstwo, four, or eight nuclei.

48

The availability of molecular techniques has led

Table 1. Drugs for Prophylaxis against Pneumocystis

Pneumonia.

Drug Dose Route Comments

Trimethoprim–sulfamethoxazole

1 double-strength tablet daily or 1 single-strength tablet daily

1 double-strength tablet 3 times per week

Oral First choice

Alternate choice

Dapsone 50 mg twice daily or100 mg daily

Oral Ensure patient does not have glucose-6-phosphate dehydrogenase deficiency

Dapsone plus pyrimethamine plus leucovorin

50 mg daily50 mg weekly25 mg weekly

Oral

Dapsone plus pyrimethamine plus leucovorin

200 mg weekly75 mg weekly25 mg weekly

Oral

Pentamidine 300 mg monthly Aerosol

Atovaquone 1500 mg daily Oral Give with high-fat meals, for maximal absorption

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to major advances in our understanding of the biol-ogy of pneumocystis in the past several years. Keymolecules have been identified in the mitotic cellcycle, cell-wall assembly, signal-transduction cas-cades, and metabolic pathways. The use of heter-ologous fungal systems to study the expression ofpneumocystis genes has helped this effort, butstandard biochemical and genetic analysis withinthe organism will be necessary to confirm the func-tion of these molecules, once it becomes possibleto culture pneumocystis.

The first specific molecule identified from pneu-mocystis was a glycoprotein with an apparent mo-lecular mass under reducing conditions of 95 to120 kD.

49,50

Termed glycoprotein A, or major sur-face glycoprotein, this molecule is heavily glyco-sylated with mannose-containing carbohydratesand has an integral role in the attachment of pneu-mocystis to host cells.

50-54

Glycoprotein A con-sists of a mixture of proteins that are encoded by afamily of genes in pneumocystis, but only a singleglycoprotein A is expressed by pneumocystis atany one time.

55,56

This surface glycoprotein is im-munogenic and antigenically distinct in everyform of pneumocystis infecting various mamma-lian hosts.

49,57,58

The major component of the cystcell wall is beta-1,3-glucan, which is composed ofhomopolymers of glucose molecules with a beta-1,3-linked carbohydrate core and side chains ofbeta-1,6- and beta-1,4-linked glucose.

59

The cyst wall also contains chitins and other

complex polymers, including melanins. In addi-tion to providing the cell wall with stability, pneu-mocystis glucan is at least partly responsible forthe marked inflammatory response in the lungs ofthe infected host.

60,61

The pneumocystis beta-1,3-

glucan synthetase gene,

GSC1,

mediates the po-lymerization of uridine 5'-diphosphoglucose intobeta-1,3-glucan.

62

Inhibitors of beta-1,3-glucansynthetase are effective in clearing the cystic formsof pneumocystis from the lungs of infected ani-mals.

63,64

Perturbation of the pneumocystis cell-wall assembly represents an attractive target forthe treatment of pneumocystis pneumonia, becausethe biosynthetic machinery for generating glucan isnot present in mammals.

Because currently it is not possible to grow pneu-mocystis outside an infected host, the functionalanalysis of the pneumocystis cell cycle and of sig-nal-transduction molecules has been performedwith the use of heterologous expression in relatedfungi. The pneumocystis cdc2 cyclin-dependent ki-nase, cdc13 B-type cyclin, and cdc25 mitotic phos-phatase all complement the function of cell-cycleregulation in yeast that harbor mutations for thesegenes.

65-67

Fungi use mitogen-activated protein ki-nase signal-transduction cascades to regulate thecellular responses for mating, environmental stress,cell-wall integrity, and pseudohyphal or filamen-tous growth. Mitogen-activated protein kinase mol-ecules homologous to those found in mating andcell-wall-integrity pathways have been identified inpneumocystis.

68-70

The gene encoding pneumocys-tis mitogen-activated protein kinase (

PCM

) func-tionally complements pheromone signaling in

Sac-charomyces cerevisiae.

70

Furthermore, the finding ofenhanced activity of

PCM

in trophic forms as com-pared with that in cysts suggests that the trophicforms use this pathway for transitions in the life cy-cle of the organism.

70

In the cell-wall integrity path-way, the kinases Bck1 and Mpk1 function in re-sponse to elevated temperature.

68,71,72

The trophic forms of pneumocystis adheretightly to the alveolar epithelium as the infectionbecomes established. The binding of pneumocys-tis to epithelial cells in the lung activates specificsignaling pathways in the organisms, including thegene encoding PCSTE20 kinase, which signal re-sponses for mating and proliferation in fungal or-ganisms.

73

Other signaling molecules, includingthe putative pheromone receptors, heterotrimericG-protein subunits, and transcription factors, havealso been identified.

74-76

Investigators are further evaluating specificmolecules within pneumocystis as potential drugtargets. These molecules include dihydrofolatereductase, the product of which is the target of tri-methoprim; thymidylate synthase; inosine mono-

Table 2. Treatment of Pneumocystis

Pneumonia.

Drug Dose Route Comments

Trimethoprim–sulfameth-oxazole

15–20 mg/kg75–100 mg/kg

daily in divid-ed doses

Oral orintravenous

First choice

Primaquine plusclindamycin

30 mg daily600 mg three

times daily

OralAlternate

choice

Atovaquone 750 mg twotimes daily

Oral Alternate choice

Pentamidine 4 mg/kg daily 600 mg daily

IntravenousAerosol

Alternate choice

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phosphate dehydrogenase, which is inhibited bymycophenolic acid;

S

-adenosyl-

l

-methionine:ste-rol C-24 methyl transferase, which is involved inthe biosynthesis of sterol; and lanosterol 14

a

-demethylase, the target enzyme of azole antifungalcompounds.

77-80

With work on the cloning of thepneumocystis genome continuing, the identifica-tion of additional treatment targets is anticipated.

Effective inflammatory responses in the host arerequired to control pneumocystis pneumonia. Ex-uberant inflammation, however, also promotes pul-monary injury during infection. Severe pneumocys-tis pneumonia is characterized by neutrophilic lunginflammation that may result in diffuse alveolardamage, impaired gas exchange, and respiratoryfailure. Indeed, respiratory impairment and deathare more closely correlated with the degree of lunginflammation than with the organism burden inpneumonia.

9

Although patients with neutropeniaoccasionally become infected with pneumocystis,they do not appear to be inordinately predisposedto this infection as compared with other groups ofimmunosuppressed patients.

lymphocyte responses to pneumocystis

Immune responses directed against pneumocys-tis involve complex interactions between CD4+T lymphocytes, alveolar macrophages, neutrophils,and the soluble mediators that facilitate the clear-ance of the infection. In particular, the activity ofCD4+ T cells is pivotal in the host’s defenses againstpneumocystis, in both animals and humans, andthe risk of infection increases with a CD4+ count ofless than 200 per cubic millimeter.

7,81

CD4+ cellsfunction as memory cells that orchestrate the host’sinflammatory responses by means of the recruit-ment and activation of other immune effector cells,including monocytes and macrophages, to attackthe organism. Mice with severe combined immu-nodeficiency (SCID) lack functional T and B lym-phocytes, and spontaneous pneumocystis infectionmay develop in them by three weeks of age, provid-ing an excellent model for understanding the func-tion of lymphocytes in this disease.

82

SCID micehave progressive pneumocystis infection, despitethe presence of otherwise functional macrophag-es and neutrophils.

82,83

When the immune systemis reconstituted with the use of CD4+ spleen cells,

however, the mice regain the ability to clear infec-tion effectively.

84,85

The mechanisms by which CD4+ cells mediatea defense against pneumocystis have only begunto emerge in recent years. Macrophage-derived tu-mor necrosis factor

a

(TNF-

a

) and interleukin-1are believed to be necessary for initiating pulmo-

host response to pneumocystis infection

Figure 3. Proposed Pneumocystis Life Cycle.

The life cycle of pneumocystis is complex, and several forms are seen during infection. The electron micrograph in Panel A shows a trophic form that is tightly adherent to the alveolar epithelium by apposition of its cell membrane with that of the host lung cell membrane. During infection, trophic forms are more abundant than cysts (approximately 9:1), and the majority of the trophic forms are believed to be haploid during normal growth, with a smaller fraction that are diploid. Trophic forms attach to one another, as shown in the electron micrograph in Panel B, and clusters of clumped trophic forms can be seen during infection. The events that lead to the formation of the cyst, shown in the electron micrograph in Panel C, are unclear, but we hypothesize that the trophic forms conjugate and mature into cysts, which contain two, four, or eight nuclei as they mature.

ProposedPneumocystis

Life Cycle

Mitosis

Cyst forms

Meiosisand

mitosis

C

A

B

A B C

Type Ialveolar cell

Trophicforms

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nary responses to pneumocystis infection that aremediated by CD4+ cells. The cells proliferate in re-sponse to pneumocystis antigens and generate cy-tokine mediators, including lymphotactin and in-terferon gamma.

85

Lymphotactin, a chemokine,acts as a potent chemoattractant for further lym-phocyte recruitment in pneumocystis pneumonia.

86

Interferon gamma strongly activates the macro-phage production of TNF-

a

, superoxides, and reac-tive nitrogen species, each of which is implicated inthe host defense against pneumocystis.

87,88

Aero-solized interferon gamma reduces the intensity ofinfection in rats infected with pneumocystis, regard-less of the degree of CD4+ depletion.

87

Although T lymphocytes are essential for theclearance of pneumocystis, data suggest that T-cellresponses may also result in substantial pulmo-nary impairment during pneumonia. For instance,in SCID mice infected with pneumocystis, normaloxygenation and lung function occur despite activeinfection until the late stages of the disease.

83

Whenthe immune systems in these animals are reconsti-tuted with the use of intact spleen cells, an intenseT-cell–mediated inflammatory response ensues, re-sulting in substantially impaired gas exchange.

83

In the absence of brisk lung inflammation, pneu-mocystis has little direct effect on pulmonary func-tion. In a similar manner, in patients who have un-dergone bone marrow transplantation the clinicalonset of pneumocystis pneumonia and of mostmarked alterations in lung function occur duringengraftment.

89

Pneumocystis pneumonia also re-sults in the marked accumulation of CD8+ T lym-phocytes in the lung.

90

macrophages in host defenseagainst pneumocystis

Alveolar macrophages are the principal phagocytesmediating the uptake and degradation of organ-isms in the lung. When there are no opsonins inthe epithelial-lining fluid, the uptake of pneumocys-tis is mediated mainly through the macrophagemannose receptors, pattern-recognition moleculesthat interact with the surface mannoprotein, gly-coprotein A.

52,91

After they have been taken up bymacrophages, pneumocystis organisms are incor-porated into phagolysosomes and degraded.

92

The function of macrophages is impaired inpatients with AIDS, malignant disease, or both, re-sulting in the reduced clearance of pneumocystis.

93

In macrophage-depleted animals the resolutionof pneumocystis infection is impaired.92 Macro-

phages produce a large variety of proinflammatorycytokines, chemokines, and eicosanoid metabolitesin response to phagocytosis of pneumocystis.92

Although these mediators participate in eradicat-ing pneumocystis, they also promote pulmonaryinjury.

cytokine and chemokine networks TNF-a has important effects during pneumocys-tis pneumonia.94,95 The clearance of pneumocys-tis is delayed when TNF-a is neutralized by anti-bodies or inhibitors in animals with pneumocystispneumonia.95,96 TNF-a promotes the recruitmentof neutrophils, lymphocytes, and monocytes. Al-though their recruitment is important for clearanceof the organisms, these cells injure the lung by re-leasing oxidants, cationic proteins, and proteases.TNF-a also induces the production of other cyto-kines and chemokines, including interleukin-8 andinterferon gamma, which stimulate the recruitmentand activation of inflammatory cells during pneu-mocystis pneumonia.

The cell wall of pneumocystis contains abun-dant beta-glucans, and studies have confirmed thatthe production of TNF-a by alveolar macrophagesis mediated by recognition of the beta-glucan com-ponents of pneumocystis.60 Macrophages displayseveral potential receptors for glucans, includingCD11b/CD18 integrin (CR3), dectin-1, and toll-likereceptor 2.97,98 The activation of macrophages bypneumocystis is augmented by proteins in the hostsuch as vitronectin and fibronectin that bind theglucan components on the organism.99

The cysteine–X amino acid–cysteine chemo-kines, such as interleukin-8, macrophage-inflam-matory protein 2, and the interferon-inducible pro-tein of 10 kD, which are potent chemoattractantsfor neutrophils, are important during pneumocys-tis infection. Interleukin-8 is correlated with bothneutrophil infiltration of the lung and impaired gasexchange during severe pneumocystis pneumonia.Furthermore, the level of interleukin-8 in broncho-alveolar-lavage fluid may serve as a predictor ofsevere respiratory compromise and death from thedisease.100 Isolated pneumocystis beta-glucan stim-ulates alveolar macrophages and alveolar epithe-lial cells to produce marked quantities of macro-phage inflammatory protein 2.60,61 The neutrophilsrecruited into the lungs release reactive oxidantspecies, proteases, and cationic proteins, which di-rectly injure capillary endothelial cells and alveolarepithelial cells.

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alveolar epithelial cells and proteinsTrophic forms adhere tightly to type I alveolar cellsthrough interdigitation of their membranes withthose of the host.101 The binding of pneumocystisto the epithelium is facilitated by interactions ofproteins in the host, such as fibronectin and vit-ronectin, that bind to the surface of pneumocystisand mediate the attachment to integrin receptorspresent on the alveolar epithelium.102 In infectedtissues, type I alveolar cells with adherent pneu-mocystis appear vacuolated and eroded.103 How-ever, studies of cultured lung epithelial cells haveshown that the adherence of pneumocystis alonedoes not disrupt the structure or barrier function ofalveolar epithelial cells, though proliferative repairof the epithelium is reduced.104,105 It is thereforeunlikely that the adherence of pneumocystis to al-veolar epithelium is by itself responsible for the dif-fuse alveolar damage in severe pneumonia. Rather,the inflammatory responses in the host are primar-ily responsible for the compromise of the alveolar-capillary surface.

Electron microscopical studies have shown thatpneumocystis organisms are embedded in protein-rich alveolar exudates, which contain abundant fi-bronectin, vitronectin, and surfactant proteins Aand D.106,107 In contrast, surfactant protein B is re-duced during pneumonia.108 Both surfactant pro-tein A and surfactant protein D interact with theglycoprotein A components of the surface of pneu-mocystis.52,106 Surfactant protein A modulates theinteractions of pneumocystis with the alveolar mac-rophages.109,110 In contrast, surfactant protein Dmediates the aggregation of the pneumocystis or-ganisms,111 but because the aggregated organismsare extremely poorly taken up by macrophages,many organisms may escape elimination.111 Pul-

monary surfactant phospholipids, which contrib-ute to the low surface tension in the alveoli, arereduced during pneumocystis pneumonia, and ab-normalities in the composition and function of thesurfactant are the result of the host’s inflammatoryresponse to pneumocystis, rather than direct ef-fects of the organisms on the surfactant compo-nents.112

Pneumocystis pneumonia remains a serious causeof sickness and death in immunocompromised pa-tients. The epidemiology of this infection is only be-ginning to emerge, but it includes the transmissionof organisms between susceptible hosts as well asprobable acquisition from environmental sources.Lung injury and respiratory impairment duringpneumocystis pneumonia are mediated by markedinflammatory responses in the host to the organ-ism. Trimethoprim–sulfamethoxazole with adjunc-tive corticosteroid therapy to suppress lung inflam-mation in patients with severe infection remainsthe preferred treatment. However, accumulating ev-idence of mutations of the gene that encodes dihy-dropteroate synthase in pneumocystis has arousedconcern about the potential for the emergence ofresistance to sulfa agents, which have been themainstay of prophylaxis and treatment of pneu-mocystis pneumonia. An improved understandingof the basic biology of pneumocystis has helped todefine new targets for the development of drugsto treat this important infection.

Supported by grants (R01 AI-48409 to Dr. Thomas and R01 HL62150 and R01 HL 55934 to Dr. Limper) from the National Insti-tutes of Health.

We are indebted to Pawan K. Vohra, Robert Vassallo, and The-odore J. Kottom for many helpful discussions.

summary

references

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